PROJECT SUMMARY Faithful replication of DNA and repair of damaged DNA, essential to cell propagation and survival, proceeds via the action of multi-protein machines. While considerable progress has been made in elucidating the mechanisms of DNA replication and repair from studies of bacteria and other lower organisms, information on humans is lacking because the protein sequences and structures are not conserved. Our long-term goals are to understand the action of human DNA replication and nucleotide excision repair (NER) machinery. Our strategy is to elucidate the structural basis for these processes using the full complement of modern structural biology tools and interpret this information using a range of biochemical and biological approaches. We have shown in the SV40 model system that the helicase that creates the single-stranded DNA (ssDNA) at the origin of replication also actively loads the human ssDNA binding protein replication protein A (RPA), and that this action is essential for DNA priming by DNA polymerase ?-primase (pol-prim). We have also characterized the complex and dynamic structural architectural of the modular RPA protein as it engages this ssDNA template and have begun to address the quandary of how RPA releases the template to hand off to pol-prim. Once the primase subunits are loaded on the DNA template, an ~10 nt RNA is synthesized and the primed template is transferred to the pol ? subunits for extension to the final ~30 nt of RNA-DNA primer. Our goal is to continue to define the trajectory of actions on the template as it is replicated. The next phase of our research is to solve the fundamental mysteries about the release of RPA and loading of pol-prim onto the template, the counting of primer length by primase, and the hand-offs from primase to pol ? and in turn to pol ? or pol ?. Our NER research seeks to understand the mechanism of action of the human NER machinery, discern the biochemical bases for malfunctions caused by disease-associated mutations, and ultimately use this knowledge to help evaluate the potential of NER-targeted chemotherapies. NER requires the assembly of >20 proteins around the lesion to create a platform for positioning the two nucleases that cut out the damaged DNA. We have defined critical DNA binding characteristics of two core NER factors: XPA and XPC. Our current focus is on the scaffold that directs the assembly and spatial organization of this ?pre-incision? complex (PIC), which involves the dual action of XPA and RPA. The goal is to obtain structural understanding and functional validation of the interactions between XPA and RPA and their role as the PIC scaffold by: (i) determining the structure of XPA and RPA bound to a NER model substrate; (ii) investigating structure-based and disease- associated mutations in XPA in cell-based assays of the repair of DNA lesions and co-localization with other NER factors. From this scaffold, we will assemble and characterize relevant sub-complexes along the trajectory to the complete NER PIC.